Identification of the first Alu-mediated gross deletion involving the BCKDHA gene in a compound heterozygous patient with maple syrup urine disease.

AIMS: To investigate a family with clinical symptoms of maple syrup urine disease and reveal a genetic cause underlying this disease. METHODS: Targeted capture sequencing was used to screen for mutations in the patient. Real-Time PCR was carried out to perform exon 1, 5, 9 CNV analysis of samples from the patient's father, mother and sister. Whole genome sequencing was performed to map the approximate location of the break points of the gross deletion. Long-range PCR and Sanger sequencing were performed to identify the length of the deletion and to locate the break points. RESULTS: The patient is a compound heterozygous mutation including a small deletion mutation (c.1227_1229del chr19: 41930402) and a gross novel deletion including exon1-9 in BCKDHA. The junction site of the gross deletion was localized within a microhomologous sequence in two Alu elements. CONCLUSIONS: This study is the first time report on rearrangement sequences in BCKDHA mediated by Alu element, which resulted in MSUD. Our results may also offer new insights into the formation and pathogenicity of MSUD, and may be useful to genetic counseling and genetic testing.

The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.

Essential roles of S100A10 in Toll-like receptor signaling and immunity to infection.

Toll-like receptors (TLRs) are key pattern recognition receptors that mediate innate immune responses to infection. However, uncontrolled TLR activation can lead to severe inflammatory disorders such as septic shock. The molecular mechanisms through which TLR responses are regulated are not fully understood. Here, we demonstrate an essential function of S100A10 in TLR signaling. S100A10 was constitutively expressed in macrophages, but was significantly downregulated upon TLR activation. S100A10-deficient macrophages were hyperresponsive to TLR stimulation, and S100A10-deficient mice were more sensitive to endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Mechanistically, S100A10 regulated macrophage inflammatory responses by interfering with the appropriate recruitment and activation of the receptor-proximal signaling components and eventually inhibited TLR-triggered downstream signaling. These findings expand our understanding of TLR signaling and establish S100A10 as an essential negative regulator of TLR function and a potential therapeutic target for treating inflammatory diseases.

EVI1 induces autophagy to promote drug resistance via regulation of ATG7 expression in leukemia cells.

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor, which is abnormally expressed in myeloid leukemia and other several solid cancers. It is associated with short survival as well as anticancer drug resistance. Autophagy is a protective mechanism that promotes cancer cell growth and survival under stressed conditions including clinical drug treatment. Here evidences are provided that EVI1 induces autophagy and mediated drug resistance in myeloid leukemia cells. Both knockdown using RNAi and pharmacological inhibition of autophagy significantly increase sensitivity to cytotoxic drug treatment in EVI1high cells. Mechanistic studies revealed that EVI1 regulated autophagy by directly binding to autophagy-related gene autophagy related 7 (ATG7) promoter and transcriptionally upregulating its expression. Notably, ATG7 expression was positively correlated with EVI1 in bone marrow mononuclear cells from myeloid leukemia patients. Acute myeloid leukemia patients with high level of EVI1 are associated with unfavorable overall survival, which was aggravated by simultaneous high expression of ATG7 in these patients. Furthermore, ChIP and firefly luciferase reporter assay identified an EVI1-binding site at 227 upstream promoter region of ATG7 which regulated its transcription. In addition, enforced expression of EVI1 also increased intracellular reactive oxygen species and ATG7 mRNA levels as well as autophagy activity, whereas the increase was attenuated after treatment with reactive oxygen species scavenger, suggesting the involvement of reactive oxygen species in EVI1-induced autophagy. These findings demonstrate that EVI protects myeloid leukemia cell from anticancer drug treatment by inducing autophagy through dual control of ATG7. These results might present a new therapeutic approach for improving treatment outcome in myelogenous leukemia with EVI1high.

BCL3 Expression Is a Potential Prognostic and Predictive Biomarker in Acute Myeloid Leukemia of FAB Subtype M2.

Although the implication of BCL3 has been disclosed in human chronic lymphocytic leukemia as well as other solid tumors, the diagnostic and prognostic of BCL3 expression in acute myeloid leukemia (AML) remains largely unclear. In this study, we isolated total RNA from bone marrow mononuclear cells collected from 101 de novo AML patients and 27 healthy donors. After reverse transcription, quantitative real-time PCR was performed to detect BCL3 expression level. BCL3 mRNA level was significantly down-regulated in BMMCs of AML patients compared with healthy controls (P = 0.0015). BCL3 was showed a higher level in AML patients with poor-risk karyotypes than that of in patients with favorable/intermediate-risk karyotypes (P = 0.014). ROC analysis demonstrated that BCL3 could effectively differentiate AML patients from normal controls. Among the French-American-British (FAB) subtypes, the frequency of low BCL3 expression in M2 subtypes is significantly higher than that of in the other subtypes M1/M4/M5/M6/M7 (P = 0.006), and mildly lower in myelomonocytic/monocytic subtypes M4/M5 (P = 0.064) than those in M1/M2/M6/M7 subtypes. Chromosome analysis revealed that BCL3low patients had a remarkably higher frequency of t (8;21) abnormality (P = 0.0047) and lower frequency of normal karyotype (P = 0.0059) than BCL3high patients. BCL3high patients showed a significantly higher frequency of FLT3-ITD mutation (P = 0.028) and lower frequency of C-Kit mutation (P = 0.0232) than BCL3low patients. Although there were no significant differences in complete remission and overall survival between BCL3low and BCL3high groups, patients with high BCL3 expression markedly shorter overall survival (OS, P = 0.049), relapse-free survival (RFS, P = 0.027) and disease-free survival (DFS, P = 0.042) in M2 AML than low BCL3 expression patients. Additionally, in AMLs of M2 subtype, high BCL3 expression patients had markedly lower complete remission (CR) rate (P = 0.0317) after the second induction treatment than patients with BCL3 low expression. Thus, these findings indicated that BCL3 appeared as a promising molecular biomarker of pediatric acute myeloid leukemia with unfavorable prognosis.

High level of miR-196b at newly diagnosed pediatric acute myeloid leukemia predicts a poor outcome.

Differential expression of microRNAs (miRNAs) has been implicated in leukemogenesis. We investigate the expression pattern of miR-196b. Using quantitative real-time PCR (qRT-PCR), we detected the expression of miR-196b and its correlated genes (SMC1A/MLH1) in initial pediatric AML. A significant association was observed between overexpression of miR-196b and inferior overall survival of pediatric AML (Log Rank P<0.0001). AML M4/5 subtype, high white blood cell (WBC) count at presentation, MLL rearrangement, or FLT3-ITD mutation at diagnosis and non-remission group after the first induction chemotherapy possessed higher miR-196b expression. Furthermore, a positive relationship was found between the expression of miR-196b and SMC1A/MLH1 (Spearman's r=0.37 and 0.44, P=0.001 and <0.0001, respectively). Taken together, these findings suggest that differentially high expression of miR-196b in diagnostic marrow samples of pediatric AML is associated with unfavorable outcome, and miR-196b potentially can be a novel biomarker for the diagnosis, prognosis and treatment in pediatric AML.

Rapamycin inhibits pre-B acute lymphoblastic leukemia cells by downregulating DNA and RNA polymerases.

Rapamycin has been shown to inhibit the growth of leukemic cells via an unknown mechanism. In our current study, we show that rapamycin activates autophagy in pediatric t(1;19) pre-B acute lymphoblastic leukemia (pre-B ALL) cells and thereby inhibits proliferation and induces growth arrest in these cells. Rapamycin was found to downregulate an extensive array of positive cell cycle regulators, reduce the total DNA and RNA levels, and specifically downregulate the gene transcription of DNA pol δ1 and RNA pol II. Furthermore, we show that both rapamycin and starvation caused a downregulation of the DNA pol δ1 and RNA pol II proteins which was reversed by the autophagy inhibitor 3-MA. Consistent with the results of our autophagic flux analysis, confocal microscopy indicated that both rapamycin and starvation cause the colocalization of DNA pol δ1 and RNA pol II with GFP-LC3 at autophagosomes. This colocalization was blocked by the autophagy inhibitor bafilomycin A1 which inhibits the fusion between autophagosomes and lysosomes. These data suggest that rapamycin inhibits the growth of pediatric t(1;19) pre-B ALL cells through both transcriptional inhibition and autophagic degradation of DNA pol δ1 and RNA pol II.

Alternative messenger RNA splicing of autophagic gene Beclin 1 in human B-cell acute lymphoblastic leukemia cells.

Beclin 1 is a key factor for initiation and regulation of autophagy, which is a cellular catabolic process involved in tumorigenesis. To investigate the role of alternative splicing of Beclin1 in the regulation of autophagy in leukemia cells, Beclin1 mRNA from 6 different types of cell lines and peripheral blood mononuclear cells from 2 healthy volunteers was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants were analyzed by DNA sequencing. A transcript variant of Beclin 1 gene carrying a deletion of exon 11, which encoded a C-terminal truncation of Beclin 1 isoform, was found. The alternative isoform was assessed by bioinformatics, immunoblotting and subcellular localization. The results showed that this variable transcript is generated by alternative 3' splicing, and its translational product displayed a reduced activity in induction of autophagy by starvation, indicating that the spliced isoform might function as a dominant negative modulator of autophagy. Our findings suggest that the alternative splicing of Beclin 1 might play important roles in leukemogenesis regulated by autophagy.

Role of autophagy in acute myeloid leukemia therapy.

Despite its dual role in determining cell fate in a wide array of solid cancer cell lines, autophagy has been robustly shown to suppress or kill acute myeloid leukemia cells via degradation of the oncogenic fusion protein that drives leukemogenesis. However, autophagy also induces the demise of acute leukemia cells that do not express the known fusion protein, though the molecular mechanism remains elusive. Nevertheless, since it can induce cooperation with apoptosis and differentiation in response to autophagic signals, autophagy can be manipulated for a better therapy on acute myeloid leukemia.

Comparative analyses by sequencing of transcriptomes during skeletal muscle development between pig breeds differing in muscle growth rate and fatness.

Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.

Lipopolysaccharide-induced miR-1224 negatively regulates tumour necrosis factor-α gene expression by modulating Sp1.

The innate immune response provides the initial defence mechanism against infection by other organisms. However, an excessive immune response will cause damage to host tissues. In an attempt to identify microRNAs (miRNAs) that regulate the innate immune response in inflammation and homeostasis, we examined the differential expression of miRNAs using microarray analysis in the spleens of mice injected intraperitoneally with lipopolysaccharide (LPS) and saline, respectively. Following challenge, we observed 19 miRNAs up-regulated (1.5-fold) in response to LPS. Among these miRNAs, miR-1224, whose expression level increased 5.7-fold 6 hr after LPS injection and 2.3-fold after 24 hr, was selected for further study. Tissue expression patterns showed that mouse miR-1224 is highly expressed in mouse spleen, kidney and lung. Transfection of miR-1224 mimics resulted in a decrease in basal tumour necrosis factor-α (TNF-α) promoter reporter gene activity and a down-regulation of LPS-induced TNF-α mRNA in RAW264.7 cells. With public databases of miRNA target prediction, miR-1224 was shown to bind to the 3' untranslated region (UTR) of Sp1 mRNA, whose coding product controls TNF-α expression at the transcriptional level. Furthermore, we found that in HEK-293 cells, the activity of the luciferase reporter bearing Sp1 mRNA 3' UTR was down-regulated significantly when transfected with miR-1224 mimics. After transfection of miR-1224 in RAW264.7 cells, nucleus Sp1 protein level decreased, and when endogenous miR-1224 was blocked, the decrease was abolished. Therefore, we initially speculated that miR-1224 was a negative regulator of TNF-α in an Sp1-dependent manner, which was confirmed in vivo by chromatin immunoprecipitation assay, and might be involved in regulating the LPS-mediated inflammatory responses.

Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling.

BACKGROUND: There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology. RESULTS: H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity. CONCLUSIONS: The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.

Characterization and transcriptional regulation analysis of the porcine TNFAIP8L2 gene.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a new member of the tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family that functions as an important factor in the maintenance of immune homeostasis. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine TNFAIP8L2. RH mapping using the IMpRH panel showed that this gene was closely linked to microsatellite marker SW512 in pig chromosome 4. Subcellular localization analysis showed GFP-TNFAIP8L2 fusion protein distributed in nucleus as well as cytoplasm including mitochondria and endoplasmic reticulum. Real-time PCR analysis revealed that porcine TNFAIP8L2 was more highly expressed in spleen than other tissues. To understand its characterization of transcriptional regulation, we cloned approximately 2 kb of 5'-regulatory region upstream to the porcine TNFAIP8L2 translational start site and generated sequential deletion constructs evaluated in dual-luciferase reporter assay. The results demonstrated that its core promoter is 435 base pairs (bp) upstream to the transcription initiation site. Then, site-directed mutation experiment combined with electrophoretic mobility shift assay (EMSA) indicated that M-CAT binding factor (MCBF) and activator protein 1 (AP-1) were important transcription factors for porcine TNFAIP8L2. These findings provide an important basis for further understanding of porcine TNFAIP8L2 regulation and function in swine.

Understanding PRRSV infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing.

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.

Proteome changes of lungs artificially infected with H-PRRSV and N-PRRSV by two-dimensional fluorescence difference gel electrophoresis.

BACKGROUND: Porcine reproductive and respiratory syndrome with PRRS virus (PRRSV) infection, which causes significant economic losses annually, is one of the most economically important diseases affecting swine industry worldwide. In 2006 and 2007, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) happened in China and Vietnam. However little data is available on global host response to PRRSV infection at the protein level, and similar approaches looking at mRNA is problematic since mRNA levels do not necessarily predict protein levels. In order to improve the knowledge of host response and viral pathogenesis of highly virulent Chinese-type PRRSV (H-PRRSV) and Non-high-pathogenic North American-type PRRSV strains (N-PRRSV), we analyzed the protein expression changes of H-PRRSV and N-PRRSV infected lungs compared with those of uninfected negative control, and identified a series of proteins related to host response and viral pathogenesis. RESULTS: According to differential proteomes of porcine lungs infected with H-PRRSV, N-PRRSV and uninfected negative control at different time points using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry identification, 45 differentially expressed proteins (DEPs) were identified. These proteins were mostly related to cytoskeleton, stress response and oxidation reduction or metabolism. In the protein interaction network constructed based on DEPs from lungs infected with H-PRRSV, HSPA8, ARHGAP29 and NDUFS1 belonged to the most central proteins, whereas DDAH2, HSPB1 and FLNA corresponded to the most central proteins in those of N-PRRSV infected. CONCLUSIONS: Our study is the first attempt to provide the complex picture of pulmonary protein expression during H-PRRSV and N-PRRSV infection under the in vivo environment using 2D-DIGE technology and bioinformatics tools, provides large scale valuable information for better understanding host proteins-virus interactions of these two PRRSV strains.

A deep investigation into the adipogenesis mechanism: profile of microRNAs regulating adipogenesis by modulating the canonical Wnt/beta-catenin signaling pathway.

BACKGROUND: MicroRNAs (miRNAs) are a large class of tiny non-coding RNAs (approximately 22-24 nt) that regulate diverse biological processes at the posttranscriptional level by controlling mRNA stability or translation. As a molecular switch, the canonical Wnt/beta-catenin signaling pathway should be suppressed during the adipogenesis; However, activation of this pathway leads to the inhibition of lipid depots formation. The aim of our studies was to identify miRNAs that might be involved in adipogenesis by modulating WNT signaling pathway. Here we established two types of cell model, activation and repression of WNT signaling, and investigated the expression profile of microRNAs using microarray assay. RESULTS: The high throughput microarray data revealed 18 miRNAs that might promote adipogenesis by repressing WNT signaling: miR-210, miR-148a, miR-194, miR-322 etc. Meanwhile, we also identified 29 miRNAs that might have negative effect on adipogenesis by activating WNT signaling: miR-344, miR-27 and miR-181 etc. The targets of these miRNAs were also analysed by bioinformatics. To validate the predicted targets and the potential functions of these identified miRNAs, the mimics of miR-210 were transfected into 3T3-L1 cells and enlarged cells with distinct lipid droplets were observed; Meanwhile, transfection with the inhibitor of miR-210 could markedly decrease differentiation-specific factors at the transcription level, which suggested the specific role of miR-210 in promoting adipogenesis. Tcf7l2, the predicted target of miR-210, is a transcription factor triggering the downstream responsive genes of WNT signaling, was blocked at transcription level. Furthermore, the activity of luciferase reporter bearing Tcf7l2 mRNA 3' UTR was decreased after co-transfection with miR-210 in HEK-293FT cells. Last but not least, the protein expression level of beta-catenin was increased in the lithium (LiCl) treated 3T3-L1 cells after transfection with miR-210. These findings suggested that miR-210 could promote adipogenesis by repressing WNT signaling through targeting Tcf7l2. CONCLUSIONS: The results suggest the presence of miRNAs in two cell models, providing insights into WNT pathway-specific miRNAs that can be further characterized for their potential roles in adipogenesis. To our knowledge, present study represents the first attempt to unveil the profile of miRNAs involved in adipogenesis by modulating WNT signaling pathway, which contributed to deeper investigation of the mechanism of adipogenesis.

Molecular cloning, expression patterns and subcellular localization of porcine TMCO1 gene.

The product of transmembrane and coiled-coil domains 1 (TMCO1) gene is a member of DUF841 superfamily of several eukaryotic proteins with unknown function. The partial DNA sequence of porcine TMCO1 was first cloned with a pig 567 bp ORF encoding 188 amino acids. By tissues expression analysis, the TMCO1 was found highly expressed in the liver, kidney and heart. The porcine TMCO1 protein was subsequently demonstrated to localize in the mitochondrion by confocal fluorescence microscopy. This data provides an important basis for conducing further studies on the functions and regulatory mechanisms underlying the role of TMCO1 gene.